Biochemical regulation of breast cancer cell expression of S1P2 (Edg-5) and S1P3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.     

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.     

Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.     

Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu-opioid receptors from rat brain: coimmunoprecipitation of the G proteins G(alpha o), G(alpha i1), and G(alpha i3)

Antibodies directed against the C-terminal and the N-terminal regions of the mu-opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the mu receptor. Two fusion proteins were constructed: One contained the 50 C-terminal amino acids of the mu receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating approximately 70% of solubilized rat brain mu receptors as determined by [3H][D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to mu receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity-purified antibody was screened for the presence of G protein a subunits, it was determined that G(alpha)o, G(alpha)i1, G(alpha)i3, and to a lesser extent G(alpha)i2, but not G(alpha)s or G(alpha)q11, were coimmunoprecipitated with the mu receptor. Inclusion of GTPgammaS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.     

Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDG-1, EDG-3, and EDG-5.

Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.     

Sphingosine 1-phosphate activates nuclear factor-kappa B through Edg receptors. Activation through Edg-3 and Edg-5, but not Edg-1, in human embryonic kidney 293 cells.

Sphingosine 1-phosphate (S1P) exerts a variety of actions as a second messenger or as an agonist that binds to one or more members of the Edg family of G protein-coupled receptors. By using human embryonic kidney 293 cells, we show that S1P activates nuclear factor-kappa B (NF-kappa B) in a receptor-dependent fashion. Edg-3 and Edg-5, which are coupled to G(i), G(q), and G(13), affect activation of NF-kappa B, whereas Edg-1, which is coupled to G(i) alone, does not. We find that the activation of NF-kappa B requires protein kinase C and Ca(2+), probably downstream of G(q), but that the activation of Rho alone by S1P, whether through G(q) or G(13), does not translate into the activation of NF-kappa B. G beta gamma has little effect of its own but potentiates the activation of NF-kappa B achieved through other G proteins. We conclude that the activation of NF-kappa B by S1P is a receptor-mediated process that relies primarily on the activation of a phospholipase C by G(q) and secondarily on effector regulation through other G proteins.     

The sphingosine 1-phosphate receptor S1P4 regulates cell shape and motility via coupling to Gi and G12/13

Sphingosine 1-phosphate (S1P) receptors represent a novel subfamily of G-protein-coupled receptors binding S1P specifically and with high affinity. Although their in vivo functions remain largely unknown, in vitro extracellular application of S1P induces distinct S1P receptor-dependent cellular responses including proliferation, differentiation, and migration. We have analyzed signaling pathways engaged by S1P(4), which is highly expressed in the lymphoid system. Here we show that S1P(4) couples directly to Galpha(i) and even more effectively to Galpha(12/13)-subunits of trimeric G-proteins, but not to Galpha(q) unlike other S1P receptors. Consequently, CHO-K1 cells ectopically expressing S1P(4) potently activate the small GTPase Rho and undergo cytoskeletal rearrangements, inducing peripheral stress fiber formation and cell rounding, upon S1P stimulation. Overexpression of S1P(4) in Jurkat T cells induces pertussis toxin-sensitive cell motility even in the absence of exogenously added S1P. In addition, S1P(4) is internalized upon binding of S1P. The capacity of S1P(4) to mediate cellular responses, such as motility and shape change through Galpha(i)- and Galpha(12/13)-coupled signaling pathways may be important for its in vivo function which is currently under investigation.     

Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop

Protease-activated receptor 1 (PAR1) is an unusual GPCR that interacts with multiple G protein subfamilies (G(q/11), G(i/o), and G(12/13)) and their linked signaling pathways to regulate a broad range of pathophysiological processes. However, the molecular mechanisms whereby PAR1 interacts with multiple G proteins are not well understood. Whether PAR1 interacts with various G proteins at the same, different, or overlapping binding sites is not known. Here we investigated the functional and specific binding interactions between PAR1 and representative members of the G(q/11), G(i/o), and G(12/13) subfamilies. We report that G(q/11) physically and functionally interacts with specific amino acids within the second intracellular (i2) loop of PAR1. We identified five amino acids within the PAR1 i2 loop that, when mutated individually, each markedly reduced PAR1 activation of linked inositol phosphate formation in transfected COS-7 cells (functional PAR1-null cells). Among these mutations, only R205A completely abolished direct G(q/11) binding to PAR1 and also PAR1-directed inositol phosphate and calcium mobilization in COS-7 cells and PAR1-/- primary astrocytes. In stark contrast, none of the PAR1 i2 loop mutations disrupted direct PAR1 binding to either G(o) or G(12), or their functional coupling to linked pertussis toxin-sensitive ERK phosphorylation and C3 toxin-sensitive Rho activation, respectively. In astrocytes, our findings suggest that PAR1-directed calcium signaling involves a newly appreciated G(q/11)-PLCε pathway. In summary, we have identified key molecular determinants for PAR1 interactions with G(q/11), and our findings support a model where G(q/11), G(i/o) or G(12/13) each bind to distinct sites within the cytoplasmic regions of PAR1.     

Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G(i), G(q), G(s), and G(betagamma)) target discrete cell surface microdomains. G(q) specifically concentrates in caveolae, whereas G(i) and G(s) concentrate much more in lipid rafts marked by GPI-anchored proteins (5' nucleotidase and folate receptor). G(q), apparently without G(betagamma) subunits, stably associates with plasmalemmal and cytosolic caveolin. G(i) and G(s) interact with G(betagamma) subunits but not caveolin. G(i) and G(s), unlike G(q), readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G(q) preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G(q), G(i), and G(s) all concentrate in lipid rafts as a complex with G(betagamma). Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G(q)-caveolin complex without G(betagamma) for trafficking, signaling, and mechanotransduction are discussed.     

The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor

Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions.     

Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides

A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]gamma-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]gamma-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor-G protein interactions.     

Light-sensitive coupling of rhodopsin and melanopsin to G(i/o) and G(q) signal transduction in Caenorhabditis elegans

Activation of G-protein-coupled receptors (GPCRs) initiates signal transduction cascades that affect many physiological responses. The worm Caenorhabditis elegans expresses >1000 of these receptors along with their cognate heterotrimeric G proteins. Here, we report properties of 9-cis-retinal regenerated bovine opsin [(b)isoRho] and human melanopsin [(h)Mo], two light-activated, heterologously expressed GPCRs in the nervous system of C. elegans with various genetically engineered alterations. Profound transient photoactivation of G(i/o) signaling by (b)isoRho led to a sudden and transient loss of worm motility dependent on cyclic adenosine monophosphate, whereas transient photoactivation of G(q) signaling by (h)Mo enhanced worm locomotion dependent on phospholipase Cβ. These transgenic C. elegans models provide a unique way to study the consequences of G(i/o) and G(q) signaling in vivo with temporal and spatial precision and, by analogy, their relationship to human neuromotor function.     

Regulation of the extracellular signal-regulated kinase pathway in adult myocardium: differential roles of G(q/11), Gi and G(12/13) proteins in signalling by alpha1-adrenergic, endothelin-1 and thrombin-sensitive protease-activated receptors

Using adenoviruses encoding RGS2, RGS4 and Lsc (regulator of G protein signalling (RGS) domain of p115 RhoGEF), we investigated the contributions of G(q/11), Gi and G(12/13) proteins to G protein-coupled receptor (GPCR)-mediated activation of the extracellular signal-regulated kinase (ERK) pathway in adult rat ventricular myocytes (ARVM). Exposure to phenylephrine, endothelin-1 (ET-1) or thrombin induced significant activation of ERK1/2 and their downstream target 90 kDa ribosomal S6 kinase (p90RSK), which was abolished by overexpression of RGS4 (inhibits signalling via G(q/11) and Gi) or RGS2 (inhibits signalling via G(q/11)). Pertussis toxin (inhibits signalling via Gi) only partially attenuated the activation of ERK1/2 and p90(RSK) by phenylephrine and ET-1, but abolished such activation by thrombin. Overexpression of Lsc (inhibits signalling via G(12/13)) did not affect the responses to phenylephrine and ET-1, but suppressed the activation of ERK1/2 and p90RSK by thrombin. We conclude that full activation of the ERK pathway in ARVM by alpha1-adrenergic, ET-1 and thrombin receptors requires the activation of distinct families of heterotrimeric G proteins.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.     

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.